THE GLUT4 GLUCOSE-TRANSPORTER AND THE ALPHA(2) SUBUNIT OF THE NA-ATPASE DO NOT LOCALIZE TO THE SAME INTRACELLULAR VESICLES IN RAT SKELETAL-MUSCLE(,K+)

The GLUT4 glucose transporter and the alpha(2) subunit of the Na+,K+-A TPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate aft er subcellular fractionation of unstimulated rat hindlimb muscles, the y do not share the same intracellular residence inside the muscle fibr e. By immunogold single- and double-labeling on ultrathin muscle cryos ections with specific antibodies, the GLUT4 glucose transporter and th e Na+,K+-ATPase alpha(2) subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and p erinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The alpha(2) subunit of the Na+,K+- ATPase was observed at the plasma membrane and in distinct subsarcolem mal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K+-ATPase alpha(2) subunit revealed that less than 6% of the two proteins co-localize in the same continuo us vesicular structures. The differential intracellular localization o f the two proteins was further confirmed by immunopurification of GLUT 4-containing membranes from muscle homogenates, in which the alpha(2) subunit of the Na+,K+-ATPase was found only at the same extent as the alpha(1) subunit of the enzyme, a protein exclusively present at the p lasma membrane.